Difference between revisions of "Team:Bielefeld-CeBiTec/Results/toolbox/photolysis"

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<p class="figure subtitle"><b>Figure 4: SDS-Page of the expressed 2-NPA-RS (left) from K2201200 with ONBY-RS from K1416000 as positive control (middle) and the basic protein expression of BL21(DE3) as negative control (right).</b><p>
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<p class="figure subtitle"><b>Figure 6: Three microwell plates tested for their suitability for the irradiation with the LED-panel. Left: Black nunc plate. Middle: Transparent nunc plate. Right: Transparent greiner plate.</b><p>
 
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Revision as of 15:23, 4 October 2017

Photolysis

Design of 2-NPA-RS

At first, we designed a gene synthesis at IDT based on the selection experiment from Peters et al. (2009) to get an aminoacyl-tRNA-synthetase to incorpate 2-nitrophenylalanine. An alignment of the protein sequences of the native M. jannaschii TyrRS which was the basis of the selection experiment and the used 2-Nitrophenylalanine-synthetase is shown in Figure 1. They differ in ten amino acids.

Figure 1: Alignment of the protein sequences of the M. jannaschii tyrosyl synthetase and the 2-Nitrophenylalanine synthetase designed by Peters et al.

Cloning of this NPA-RS in pSB1C3 and pSB3T5

We then used the protein sequence of the clone with the highest fidelity for 2-NPA and translated it into a gene sequence which was then codon optimized for E.coli. We designed it with matching overhangs of 35bp to a linearized ONBY-Part (K1416000) in pSB1C3 to get the sequence in a matching expression cassette. The psb1c3 backbone is a high copy plasmid and for an adequate usage of the aaRS it is needed on a low copy plasmid. We so used BioBrick assembly to get the insert of the new 2-NPA-Part (K2201200) in the low copy plasmid of pSB1K3 (Figure 2) for further use. The Insert of K2201200 in the low copy plasmid is available on request at the CeBiTec.

Figure 2: Two Plasmids we created for our toolkit for the iGEM community. Left: 2-NPA-RS in the pSB1C3 high copy plasmid (K2201200). Right: 2-NPA-RS in the pSB3T5 low copy plasmid (available on request) at the CeBiTec.

Design of fusion protein

We also designed two fusion proteins to verify the incorporation and functionality of the 2-NPA (Figure 3). Plasmid I (K2201320) codes for a simple GFP-streptavidin fusion protein connected by a gly-gly-ser-linker. Plasmid II (K2201321) is homologous to plasmid I but has an amber codon in the middle of the linker. If transformed in E.coli BL21(DE3) (1) only the GFP-unit will be expressed (B). If cotransfromed with an aaRS for a noncanonical amino acid but without feeding the specific ncAA (2) the aaRS will incorporate other amino acids profoundly phenylalanine in the linker (C). If cotransformed and with the 2-NPA in the culture media (3) the fusion protein will be expressed with 2-NPA in the linker (D). The fusion protein can then be irradiated by light of a wavelength of 365nm (4) to induce the cleavage of the fusion protein to its GFP-unit (E) and the streptavidin-unit (F).

Figure 3: Design of two plasmids for fusion proteins. I) Plasmid (K2201320) for reference protein of GFP (green) a linker (purple) and streptavidin (yellow) (A). II) Plasmid (K2201321) for the application protein with Amber-codon (black star) in the linker for three different protein variants after expression. 1: Solely expression leads to GFP-unit and linker to the Amber-codon (B). 2: Cotransformed with a 2-NPA-RS (K2201200) without 2-NPA leads to a fusion protein with an unspecific amino acid (presumably phenylalanine, red star) in the linker (C). 3: Cotransformed with 2-NPA-RS and 2-NPA leads to the functional fusion protein with 2-NPA (purple star) in the linker (D). 4: Irradiation of protein D leads to a cleavage of the fusion protein in the GFP-unit (E) and the streptavidin unit (F).

Proof of incorporation of AS at Amber-codon when cotransformed with NPA-RS

To proof the expression of the 2-NPA-RS in K2201200 we transformed this part, the ONBY-Part (K1416000) and a plasmid only containing the coding sequence of the fusion protein in E.coli BL21(DE3). The three transformants were cultivated in LB-Media for 16 hours at 37°C and 150 rpm. The cultures were harvested and the pellets lysed after the standard protocol of cell lysis in lysis buffer. Afterwards 15 µl of the samples were then transferred to an SDS-Page (Figure 4). There were two big bands present at approximately 35 kDA at the 2-NPA and the ONBY sample which correlates with the expected masses of the synthetases. We so are sure that the 2-NPA-RS in part K2201200 is well expressed when transformed in a matching E.coli strain like BL21(DE3).

Figure 4: SDS-Page of the expressed 2-NPA-RS (left) from K2201200 with ONBY-RS from K1416000 as positive control (middle) and the basic protein expression of BL21(DE3) as negative control (right).

To proof that our 2-NPA-RS is able to incorporate amino acids to the amber codon the parts was cotransformed with plasmid II (K2201321) in E.coli BL21(DE3) along with plasmid I (K2201320) and plasmid II was also transformed separately. After cultivation and cell lysis as mentioned above (in which one culture of the cotransformants was cultivated with 1mM and another without 2-NPA) the samples were transferred on an SDS-Page and a western-blot with anti-GFP-antibodies was performed (Figure 5).

Figure 5: Western blot with GFP-antibodies of the four different fusion proteins variants (figure 3) as proof of the functionality of the 2-NPA-RS. The band marked with a * is weak because of degradation of the fusion protein while the storage. The bands at approximately 45,0 kDa mark the mass of the whole fusion protein (~ 40,9 kDa), the bands at approximately 25,0 kDa mark the GFP-unit (~ 27,0 kDa) of the fusion protein.

Figure 5 shows that only the whole fusion protein of plasmid II was expressed (A) and only the GFP-unit of plasmid II if not cotransformed with a matching aaRS (B). The fusion protein of plasmid II was fully expressed when cotransformed with the 2-NPA-RS and when cultivated with (D) and without (C) 2-NPA in the media.

Permeability of Microwellplate by irradiation of 365nm

To proof which micro well plates are suitable for the irradiation with our LED-Panel we made a little absorption study of three plates that were available. A black and a transparent 96-well plate from nunc and a transparent well plate from greiner (Figure 6).

Figure 6: Three microwell plates tested for their suitability for the irradiation with the LED-panel. Left: Black nunc plate. Middle: Transparent nunc plate. Right: Transparent greiner plate.

The absorption of the empty plates were measured in a the Tecan Infinite M200 (Roche) at wavelengths from 300 to 450 nm (Figure 7, left). The measured adsorption values were converted to the amount of light that passes the plates in percentage (Figure 7, right). Figure 7 shows that all plates are suited for the application with the LED-panel. At 365 nm 80 % of the light permits both of the transparent plates and 87 % of the light permits the black nunc plate. A small fraction of light will always be absorbed but the amount of light that permits the plates should be high enough for the photlysis and the photoswitchung experiments.

Figure 5: Western blot with GFP-antibodies of the four different fusion proteins variants (figure 3) as proof of the functionality of the 2-NPA-RS. The band marked with a * is weak because of degradation of the fusion protein while the storage. The bands at approximately 45,0 kDa mark the mass of the whole fusion protein (~ 40,9 kDa), the bands at approximately 25,0 kDa mark the GFP-unit (~ 27,0 kDa) of the fusion protein.